Nucleic acid detection with energy transfer

ABSTRACT

PCT No. PCT/GB94/02068 Sec. 371 Date May 20, 1996 Sec. 102(e) Date May 20, 1996 PCT Filed Sep. 23, 1994 PCT Pub. No. WO95/08642 PCT Pub. Date Mar. 30, 1995A method for the detection of a nucleic acid analyte by complementary probe hybridisation and formation of a chelated lanthanide complex which, upon irradiation by light, results in a characteristic delayed luminescence emission.

A method is disclosed for the detection of analytes such as DNA or RNAsequences. The method involves forming a complex comprising the analyteand a complementary binding entity such that, for DNA or RNA,hybridisation occurs to form a duplex. Observation of the matchingcomplex is obtained by adding a third, sensitising component that eitherintercalates or groove binds with the duplex sequence (the duplexbinder). The complementary binding entity is designed to incorporate alanthanide ion and the duplex binder incorporates a ligand for thelanthanide ion that can also act as a sensitiser. Alternatively thecomplementary binding entity can be attached to the sensitiser and theduplex binder to the lanthanide ligand. When binding in thecollaborative manner is achieved, the three component system isirradiated with light which can be selectively absorbed by thesensitiser. The excited sensitiser can donate its energy to thelanthanide ion by a direct ligand to metal energy transfer mechanismsuch that the lanthanide ion becomes excited. Subsequent emission oflight from the excited lanthanide ion signals the formation of thecomplex and hence the presence of the analyte. Luminescence from thelanthanide species is characterised by a long lifetime, involving adelayed emission process, which can be measured after decay ofbackground fluorescence signals. The method may be applied to eitherheterogeneous or, preferably, homogeneous assays and may be used eitherqualitatively or quantitatively.

Methods for the in vitro detection of analytes are well known in theart. The methods include the formation of antibody-antigen complexes, asin immunoassays, and the formation of nucleic acid complexes, as inpolynucleotide hybridisation. Polynucleotide hybridisation assays usinga polynucleotide probe for verifying the presence of a targetpolynucleotide target is a well known method. Polynucleotidehybridisation is based upon the ability of a DNA or RNA sequence to formcomplexes with a complementary dna or rna strand. When single strandedpolynucleotide probes are incubated in solution with single strandedtarget molecules under defined conditions, complementary basesequences-pair to form double-stranded hybrid molecules. Suchhybridisation can occur in solution. Alternatively, either the targetstrand or the probe may be immobilised on a support in which casehybridisation leads to double stranded hybrid molecules which are thusimmobilised. In this case any unbound polynucleotide molecules may bewashed off whilst leaving the separated, immobilised duplexpolynucleotide bound to the support. See M. Grunstein and J. Wallis,Methods in Enzymology, 1979, 68, pp. 379-469; A. R. Sambrook, Methods inEnzymology, 1980, 65 (part 1), pp. 468-478; `Modified Nucleotides andMethods of Preparing and Using the Same`, by D. C. Ward, A. A. Waldropand P. R. Langer, European Patent Publication, 063,879; `DNA Probes forInfectious Disease` by A. J. Berry, and J. B. Peter, DiagnosticMedicine, 1984, March, pp. 1-8.!

The polynucleotide probes comprise a polynucleotide segment and a latentsignalling segment which is attached to the polynucleotide. Thepolynucleotide segment of the probe has the ability to hybridise (basepair) to a complementary sequence of interest within the targetpolynucleotide (the target). The latent signalling portion of the probeproduces the means by which the presence of the analyte can be verified.Whilst methods can involve, for example, fluorescence, phosphorescence,radioactivity, chromogen formation or electron density, this applicationconcerns the use of delayed luminescence.

The method for detecting the presence of a target polynucleotidegenerally involves several steps, one of which involves the separationof hybridised polynucleotide probe from the unhybridised probe ormismatched target, as in heterogeneous or sandwich type assays.Typically, double stranded polynucleotides are isolated from a samplesuspected of containing a target polynucleotide sequence. The doublestranded polynucleotides are cut into smaller segments by means ofrestriction endonuclease enzyme digestion, the segments are separated bygel electrophoresis and the segments transferred from the gel, if needsbe onto a support, for example, nitrocellulose paper.

Alternatively, the double stranded polynucleotides are either fixeddirectly onto the support without any prior enzyme digestion or takeninto a solution. If necessary the concentration of the targetpolynucleotide can be increased by use of standard amplification methodssuch as the use of the polymerase chain reaction methodology (PCR)European Patent Application 0201184; see `PCR Technology` ed. H. A.Erlich, Stockton Press, New York, 1989! before conducting thehybridisation with the probe polynucleotide in the manner describedabove. The fixed, or free polynucleotides are contacted with a solutioncontaining the polynucleotide probe and the support or solution isheated to 50°-95° C. to denature the polynucleotide double strands. Thesystem is then allowed to cool to an appropriate temperature for anappropriate time to allow hybridisation to take place. An advantage ofthe heterogeneous method using either bound target or probepolynucleotide is that, after hybridisation, the fixed hybridisedpolynucleotides can be washed to remove all unbound polynucleotides.However, a disadvantage of the heterogeneous method is that it is timeconsuming and one cannot be certain that all unbound material iscompletely removed during the washings. For this reason means foraccomplishing direct, homogeneous detection of the complementary hybridtarget to probe sequence are desirable. For homogeneous assays only asingle addition of reagents to the target polynucleotide is generallyrequired with the consequent saving of time and labour andpretreatments, such as separation, washing and electrophoretic steps,are not required before measurement of the luminescence. Furthermore,such homogeneous assays lend themselves more easily to automated methodsfor the processing of large numbers of samples.

Several methods seeking to overcome the limitations of heterogeneousassays by use of a homogeneous process have been reported (see forexample Matthews J. A. et al, Anal.Biochem., 1988, 169, 1-25). Onemethod comprises the hybridisation of two single stranded probepolynucleotides, both of which contain light-sensitive labels, with acomplementary single-stranded polynucleotide target from a sample suchthat non-radiative energy transfer occurs between the light-sensitivelabels of the first and second polynucleotide probes. At least one ofthe light sensitive labels is of the absorber-emitter type such thatenergy absorbed by this label from the emission of the otherlight-sensitive label is reemitted at a different wavelength. Thesesecondary emissions can only occur if hybridisation of both the firstand the second single-stranded polynucleotide probes to the targetpolynucleotide has taken place. The quantity of the targetpolynucleotides in the sample is related to the amount of secondarylight emitted. See European Patent Publication No. 070,685.! A drawbackof this method is that it requires two separate polynucleotide strandsto detect the presence of a target polynucleotide. Furthermore themethod requires the presence of either light sensitive probes that havecompletely different light absorbance properties, so that no directexcitation of the second label occurs and only energy transfer from thefirst to the second label is possible, or the presence ofchemiluminescent reagents as well as an absorber-emitter, emission beingpromoted by the addition of a chemiluminescent catalyst. For the lattermethod only one label can be attached per polynucleotide probe becausethe light-sensitive label is attached to the sugar moiety of a terminalnucleoside.

Another method for the detection of a target polynucleotide by means ofa homogeneous assay involves forming a hybrid between the targetpolynucleotide and the polynucleotide probe, wherein the hybrid hasbinding sites for two specific binding reagents, one of which comprisesa first label and the other a second label. The interaction of the firstand second labels provides a detectable response which is measurablydifferent when the two labelled reagents are both bound to the samehybrid, as compared to when the two labelled reagents are not so boundto the same hybrid. The formation of the hybrid assay product brings thetwo labels within approximate interaction distance of one another, e.g.as in the cases of sequential catalyst (enzyme) interaction and energytransfer. Since the labels provide a response, which is distinguishableonly when the labels are associated with a hybridised probe, noseparation step is required. See European Patent Application No.144,914.!. This method has two main embodiments. The first embodimentinvolves the generation of a component which subsequently produces acolour. This embodiment has a drawback in that it requires the use oftwo distinct chemical reactions, namely, the reaction of the first labelto produce a diffusible mediator product and the reaction of themediator product with the second label to yield a detectable product. Inaddition, detection depends on the formation and maintenance of a higherlocalised concentration of the mediator product in the vicinity of thelabel as compared to elsewhere in the solution. Furthermore, bothreactions require the use of bulky enzyme molecules attached to thepolynucleotide probe. These bulky molecules may sterically clash witheach other.

A second embodiment involves that of energy transfer, namely theemission of photons from a first label, for example, fluorescence,followed by absorption of the photons by a second label, to eitherquench the emission or to provide a second emission. This has a drawbackin that the efficiencies of such energy transfer processes are generallylow, leading to low sensitivities of the method. Furthermore, backgroundfluorescence together with adventitious excitation of the second labelby background fluorescence also detracts from the specificity of themethod.

European Patent application no. 242,527 details a through space energytransfer process; this contains as an embodiment, the use of lanthanidespecies as the energy acceptor by a through space energy couplingmechanism; it does not however foreshadow a key finding on which thisinvention is based, viz. that a direct ligand to metal charge transfermechanism can be utilised which is far more efficient in terms of energytransfer and which is far more specific in the production of theluminescence signal.

Several other related approaches to DNA recognition have been reported.Helene and colleagues use a probe DNA strand to which is attached afluorescent intercalating handle PCT Int. App. WO88,04301; C. Helene andN. T. Thuong, Pont. Acad. Sci. Scripta Varia, 1988, 70, 205-222!. Onhybridising this handle can intercalate into the adjacent duplex andthis changes the fluorescent characteristics. However this methodsuffers from lack of sensitivity since the unhybridised probe moleculesretain an incipient fluorescence characteristic and this adds to theproblems of background fluorescence interference. Barton et al. haveused a similar approach to that of Helene but use complexes of rutheniumas intercalators in place of organic fluorophores PCT Int. App.WO-88/04301; see J. K. Barton et al., Biochemistry, 1992, 31,10809-10816!. The advantage of these latter complexes is that theefficiency of luminescence is low in water but high in an intercalatingenvironment. No synergy is involved in the latter case and hence theirselectivity is not particularly high since any intercalation event leadsto luminescence.

Yet another method (European Patent Application 0382433A2) utilises apolynucleotide probe labelled with a fluorescent reagent and uses thephenomenon of fluorescence polarisation to study the binding of theprobe to the target polynucleotide.

As indicated in the above examples, fluorescence detection is widelyused in hybridisation assays. In fluorescence spectroscopy the substanceto be determined, which is present in a liquid or a solid phase, issubjected to radiation from a source of known spectral distribution, forinstance light with a limited bandwidth. The fluorescent radiationgenerated has a longer wavelength than the exciting radiation and thisradiation is specific for the substance to be determined. Themeasurement of the intensity of the fluorescent radiation constitutes aquantification of the substance to be determined. Fluorescent moietiesattached to a polynucleotide are most efficient when they have a highintensity, a relatively long emission wavelength (more than 500 nm), ahigh Stoke's shift and the ability to be bound to the polynucleotidewithout affecting its hybridisation capabilities. Aromatic agents usedin biological systems that give a strong fluorescence are well known.

Fluorescence is generally measured with a spectrofluorimeter. Adisadvantage of current methods for detecting signalling groups withspectrofluorimeters is that the detection sensitivity is limited becauseof interfering fluorescence or noise in the exciting and detectingsystems that increases the background. The background is also affectedby a heavy scattering which gives rise to an interference, especiallywhen aromatic organic agents with a small Stoke's shift (less than 50nm) are used.

Several approaches have been described that attempt to overcome thebackground problem with fluorescence detection. One approach U.S. Pat.No. 4,058,732! measures delayed luminescence, using a signalling groupthat possesses a luminescence having a much longer duration than that ofthe fluorescence of the noise and background sources. A laser pulse isused to excite the sample and the detection of the luminescence from thesignalling group is only measured after a sufficiently long time whenthe fluorescence from the noise and background sources have decayed.This method has drawbacks in that, hitherto, it is not readily adaptableto commercial use and is not amenable for a homogeneous assay.

A second approach U.S. Pat. No. 4,374,120! involves a method fordetermining the presence of an antigen by first attaching a ligand to anantibody, complexing a lanthanide metal to the ligand and then bindingthe labelled antibody to the antigen. The complex has to be separated (aheterogeneous assay) from uncomplexed antibody and the antigen-antibodycomplex then measured by transferring the lanthanide ion to a secondphotosensitising ligand. Estimation of the amount of lanthanide ion isachieved by radiating to excite the second ligand; this then transfersthe energy to the chelated metal which emits radiation at a longerwavelength and for a longer time period than the noise sources. Adrawback in this method is that it cannot be used for a homogeneousassay as well as requiring several steps.

The present invention concerns a method for the detection anddetermination of an analyte, such as a polynucleotide strand DNA, bymeans of a direct ligand-metal energy transfer system that results inthe emission of a characteristic, delayed luminescence emission.

Excitation of a sensitiser directly chelated to a lanthanide ion, suchas europium (III), is followed by the ligand-metal energy transfer.Luminescence from the metal ion is characterised by a long lifetime,which can be measured after decay of background fluorescence signals andthe light emission signals the presence of the analyte.

It is an object of this invention to provide a method for detecting ananalyte by complexing it to a binding entity to which is attached onepartner of an energy transfer system, for example a lanthanide chelatinggroup, wherein the formation of the complex allows for the localisationof a sensitising entity, which behaves as the first partner of theenergy transfer system, within a specific, binding distance of thesecond partner so that the first and second components form a closedchelated system around a lanthanide ion and, as a consequence, energyabsorbed by the first component can be directly used to excite thechelated lanthanide ion. It is a requirement of the system that thewaveband of the exciting radiation is substantially absorbed by thefirst partner of the energy transfer system and that the light emittedby the second partner, the excited lanthanide ion, is of longerwavelength than that used in the excitation step and that the durationof the emission is preferably substantially greater duration than thatof any background fluorescence or noise generated by the irradiationprocess.

The invention also comprises a method for detecting an analyte bycomplexing it to a binding entity to which is attached the first partnerof an energy transfer system, for example a sensitising entity, whereinthe formation of the complex allows for the localisation of thelanthanide chelating group, which behaves as the second partner of theenergy transfer system, within a specific binding distance of the firstpartner so that the first and second components form a closed chelatedsystem around a lanthanide ion and, as a consequence, energy absorbed bythe first component can be directly used to excite the chelatedlanthanide ion. It is a requirement of this system that the waveband ofthe exciting radiation is only substantially absorbed by the firstpartner of the ligand to metal energy transfer system and that the lightemitted by the second partner, the excited lanthanide ion, is of longerwavelength than that used in the excitation step and that preferably thelifetime of the emission is of substantially greater duration than thatof any background fluorescence or noise generated by the irradiationprocess.

This invention further provides a method for detecting the presence of atarget polynucleotide in solution by hybridising it to a polynucleotideprobe to which is attached either the first or second component of aligand-to-metal energy transfer system, comprising of a complexedlanthanide ion and a complexing sensitiser, to form a hybrid such thatthe second component of the energy transfer system may then belocalised, either by intercalation or groove binding, within a specificbinding distance of the first component to form a closed chelated systemaround the lanthanide ion and, as a consequence, energy absorbed by thefirst component can be directly used to excite the chelated lanthanideion.

The invention also comprises a method for detecting the presence of atarget polynucleotide by fixing the target polynucleotide to a support,contacting the target polynucleotide with a solution containing apolynucleotide probe to which is attached either the first or secondcomponent of an energy transfer system, comprising of a complexedlanthanide ion and a complexing sensitiser, to form a hybrid such thatthe other component of the ligand-to-metal energy transfer system maythen be localised, either by intercalation or groove binding, within aspecific binding distance of the first component to form a closedchelated system around the lanthanide ion and, as a consequence, energyabsorbed by the first component can be directly used to excite thechelated lanthanide ion.

This invention further includes a method for detecting the presence of atarget polynucleotide by fixing to a support a polynucleotide probe, towhich is attached either the first or second component of an energytransfer system, comprising of a complexed lanthanide and a complexingsensitiser, contacting the probe with a solution containing the targetpolynucleotide to form a hybrid such that the other component of theenergy transfer system may be localised, either by intercalation orgroove binding, within a specific binding distance of the firstcomponent to form a closed chelated system around the lanthanide ionand, as a consequence, energy absorbed by the first component can bedirectly used to excite the lanthanide ion.

In these assay systems the probe polynucleotide can either be completelycomplementary to the polynucleotide sequence in the target analyte, inwhich case a high level luminescence response can be obtained, or aprobe polynucleotide containing one or more mismatched bases as againstthe target sequence, in which case a reduced luminescence signal may beobtained, given appropriate polynucleotide design. In this manner onecan search for the occurrence of and site of a mutation (mutations) in atarget polynucleotide strand.

The components of the energy transfer system are a light absorbingmoiety which can act as a sensitiser. Mechanisms for such energytransfer are well documented. Singlet-singlet energy transfer can occureither by direct contact between species or by Forster energy transferprocesses in which the exchange of energy can occur through space overdistances up to 30 nm. Triplet energy transfer is more efficient bydirect contact, either by collision or by holding the partners adjacentto one another, as in metal complexes, for example by ligand-to-metalenergy transfer processes. The acceptor can be an aromatic agent or alanthanide metal. The latter process of energy transfer using alanthanide metal as the acceptor is utilised in this invention. One ofthe drawbacks with many energy transfer signalling systems is the factthat the resulting signal is emitted with a short lifetime(fluorescence) and has to be collected at the same time as backgroundsignals, the consequence being a loss of sensitivity and selectivity. Amethod to overcome this involves the use of emitters with a longlife-time, as in delayed fluorescence and phosphorescence (delayedluminescence). For such probe systems, the collection of emitted photonscan be delayed, until the fast background emission has faded, beforecollecting the delayed luminescence signal, a process called gating.Such a procedure avoids the problems associated with backgroundfluorescence and noise.

DETAILED DESCRIPTION OF THE INVENTION

1. General Description

This invention discloses a homogeneous assay for determining thepresence of a polynucleotide analyte. Homogeneous solutions include anysolution having solutes in the liquid phase. This also includessuspensions with fine suspensions or colloids or related mixtures whichare sufficiently transparent or non-scattering to enable luminescencemeasurements to be made. The homogeneous assay permits the detection ofthe analyte by a direct metal-to-ligand energy transfer process. Theassay involves hybridising the analyte with a complementary probepolynucleotide and addition of an intercalating agent to which isattached one partner of the energy transfer components. There is no needto remove unbound binding probe or unbound intercalating agent from theassay medium before detection can be achieved.

In some embodiments of the assay, all of the components are dissolved ina solution (liquid phase). In other embodiments, one, or more of thecomponents are fixed to a solid support.

The energy transfer system comprises two parts, a chelating ligand,specific for a lanthanide ion, such as the europium (III) ion and towhich the ligand is strongly bound in the range of pH values used in theassay. The ligand is selected such that the coordination sites aroundthe lanthanide ion are not completely occupied. The second component ofthe energy transfer system is a sensitising ligand, that can also bindto the lanthanide ion, whilst the latter is still bound to the otherchelating ligand. The sensitising ligand consists of an aromatic groupthat is capable of absorbing light in order to produce an excited state.The energy of this excited state is then transferred to the lanthanideion. It is a key requirement of this invention that the transfer ofexcited state energy can only occur when the sensitiser and the chelatedlanthanide ion are in close proximity, such as in direct contact byligation. Energy absorbed by the sensitiser in this two-componentcomplex (ligated europium and sensitiser) can be transmitted byspecific, local, ligand-to-metal energy transfer resulting in theformation of an excited state of the lanthanide ion. This excitedlanthanide ion may emit luminescence of a longer wavelength than thatabsorbed by the sensitiser and, in addition, the emitted fluorescence isof substantially longer duration than that of fluorescence emitted bythe sensitiser or other background fluorescence. The presence of thisdelayed luminescence indicates the presence of the analyte.

The method is applicable to the detection of polynucleotides and can becarried out in a one phase system, in a homogeneous solution assay, orin a two phase system, i.e. in a solution over a solid support. Thedetection is carried out by forming a hybridised complex between thetarget polynucleotide and a complementary probe polynucleotide to whichis affixed one of the components of the energy transfer system. Thepoint of attachment of the energy transfer component is such that itdoes not interfere with the complementarity between the targetpolynucleotide and the complementary probe. The second component of theenergy transfer system is linked to a duplex binder such as anintercalating agent or groove binder. The second component is such that,only after hybridisation between the probe polynucleotide and the targetpolynucleotide can, for example either the intercalating agent insertinto the so-formed duplex strand of polynucleotides or groove bindingoccur. For example, the intercalation allows the second component of theenergy transfer to approach the first component. Since intercalation isa reversible process, it is believed that the agent will move betweenthe various base pairs of the duplex until it is within binding distanceof the other component. This approach may also be aided by directligation of the sensitising ligand to the chelated lanthanide ion; whenthis ligation has occurred at the targeted analyte efficient energytransfer between the sensitising ligand and the chelated lanthanide ioncan take place and thus delayed luminescence be observed.

A number of embodiments are described below by way of Example.

1. The analyte is a target polynucleotide and the binding entitycomprises a complementary probe polynucleotide to which is attached achelated lanthanide ion. The sensitising ligand is either a duplexbinder or is attached to an duplex binder. All the components aredissolved in the liquid phase. By way of illustration of this embodimentsee FIG. 1.

2. The analyte is a target polynucleotide and the binding entitycomprises a complementary probe polynucleotide to which is attached asensitising ligand. The chelated lanthanide ion is attached to a duplexbinder. All the components are dissolved in the liquid phase.

3. The analyte is a target polynucleotide and is fixed onto a solidsupport. The binding entity comprises a complementary probepolynucleotide to which is attached a chelated lanthanide ion. Thesensitising ligand is either a duplex binder, or is attached to a duplexbinder. Both components of the energy transfer system are initiallydissolved in the liquid phase.

4. The analyte is a target polynucleotide and is fixed onto a solidsupport. The binding entity comprises a complementary probepolynucleotide to which is attached a sensitising ligand. The chelatedlanthanide ion is attached to a duplex binder. Both components of theenergy transfer system are initially dissolved in the liquid phase.

5. The binding entity, comprising a complementary probe polynucleotideto which is attached a chelated lanthanide ion, is fixed onto a solidsupport. The analyte is a target polynucleotide and the sensitisingligand is either a duplex binder, or is attached to a duplex binder.Both the analyte and the sensitising ligand are initially dissolved inthe liquid phase.

6. The binding entity comprising a complementary probe polynucleotide towhich is attached a sensitising ligand is fixed onto a solid support.The analyte is a target polynucleotide and the chelated lanthanide ionis attached to a duplex binder. Both the analyte and the intercalatingagent are initially dissolved in the liquid phase.

The duplex binder is an intercalator, groove binder or any other moietythat binds to duplex but not single-stranded nucleic acid. Preferably,the duplex binder is an intercalator.

Whilst we do not wish to be bound by theoretical considerations, themethod of the assay involves irradiating a sensitiser which is in directassociation with a lanthanide ion via chelation. This forms an excitedstate species of higher energy than that of the ground state. Providingthe resulting excited state has enough energy this can be transferreddirectly to the lanthanide ion, the advantage of the direct associationresulting in a very efficient energy transfer process; through spaceenergy transfer processes are generally much less efficient. The energytransfer results in the formation of an excited state lanthanide ionwhich can return to the ground state by emission of light energy. Sincethe excited state to ground state transformation of the lanthanide ionsinvolve inner shell electrons, the pattern of light emission is verycharacteristic, in which the wavelength of the emissions are not greatlyinfluenced by the local environment. The wavelength of emission is farremoved (a large Stoke's shift) from that of the initial absorptionprocess. Furthermore, the nature of the emission process results in along lifetime (delayed luminescence) which allows for the measurement ofthe emitted light after a short time interval, during which anyfluorescence emission from any background and noise processes hasdecayed. Important limitations are that the binding of the chelatingligand for the lanthanide ion should be high so that, at theconcentrations and pH employed for the assay, no, or only a very limitedamount of, dissociation of the ligand for the lanthanide occurs.Furthermore, the binding constant of the sensitising ligand for thechelated lanthanide ion should be such that, at the concentrations usedfor the assay, little random binding between these energy transferagents occurs in the absence of the target polynucleotide, since suchrandom binding will lead to energy transfer and hence the emission of abackground delayed luminescence signal.

By way of illustration, an example of a one phase assay, where theanalyte is a polynucleotide in solution, is the addition of thecomplementary polynucleotide probe, to which is attached the chelatedlanthanide ion, together with a duplex binder to which is attached asensitising ligand. The concentration of the polynucleotide probe andthe sensitising ligand is such that little random association of thesecomponents occurs. The target polynucleotide and the probepolynucleotide components are then hybridised to form a double strand ofpolynucleotide. The presence of the doubly stranded polynucleotideallows for intercalation by the sensitising reagent. The associationconstant for intercalation localises the sensitiser at the doublystranded nucleic acid and thus the local concentration of theintercalator is increased. Intercalation is a dynamicassociation--dissociation process such that movement of theintercalating agent between different intercalation sites along thematched doubly stranded polynucleotide sequence can occur. When in closeproximity to the lanthanide chelate a further binding to the metal ionby the chelating ligand can occur, thus helping to fix the components ina cooperative manner. Only when all three components, the polynucleotidetarget, the polynucleotide probe and the sensitiser come together inthis cooperative way does irradiation of the sensitiser give the delayedluminescence signal, indicating a positive identification of the target;the signal intensity can be used to quantify the analyte. Forpolynucleotide probes that do not find a complementary polynucleotidesequence, no hybridisation can occur and hence no cooperativeenhancement of the delayed luminescence would be observed.

An example of the two-phase assay would be where the polynucleotidetarget is first bound to a solid support, such as a sheet ofnitrocellulose. The assay would then be carried out by adding to thesupported polynucleotide a solution of the two reagents, the probepolynucleotide to which is attached, for example, the chelatedlanthanide sensitiser, and the intercalating reagent to which isattached the sensitising ligand. The polynucleotides are allowed tohybridise. After equilibration any excess of reagents are washed off andthe bound complex then irradiated with light and the delayedluminescence measured. If no target polynucleotide were present nointercalation could take place and hence no sensitisation of thelanthanide ion would be observed, i.e. no delayed luminescence wouldoccur.

For assays involving a polynucleotide target a complete base sequencematch of the analyte to the probe is not always essential. Thus, by wayof illustration, one could analyse for a target bearing a base mutation(or mutations) in the region complementary to the probe. This wouldcause a mismatch at this location, with the consequent formation of a`bubble` in the resulting polynucleotide duplex. From theoreticalconsiderations and/or by experimentation it is possible to design theprobe such that this bubble is located in the region where the duplexbinder-sensitizer would bind in the complex. Because intercalation, orgroove binding, would be weaker in this region, one would produce lessluminescent signal than that for the match. By comparing this with theintensity of the matching probe one would gain information on thepresence and location of the mutation(s). A further way of detectingmutations is by use of allele-specific probes designed such that themutation causes the probe not to hybridise, whilst the absence of themutation causes hybridisation and a luminescent signal. Heterozygoteswould give a reduced signal.

In a particularly convenient aspect the method of the invention is ahomogeneous, one-step assay using a PCR product as targetpolynucleotide, one or more assay components such as the complementaryprobe polynucleotide being present at the the start of the PCRamplification. Preferably all of the assay components are present. Thatis to say the method comprises a self-contained amplification anddetection system. A particular advantage is that contamination may beavoided, for example since after amplification no further reagents arerequired and therefore there is no need to unseal the reaction vessel.Examples of other convenient systems will be apparent to the molecularbiologist of ordinary skill.

Suitable hybridisation conditions for nucleic acid hybridisation may bedetermined by routine experimentation. Convenient conditions areoutlined in "Hybridisation--A Practical Approach" edited by B. D. Hamesand S. J. Higgins (IRL Press, Oxford, 1988)

2. Description of the Polynucleotide Probe Entity

This is comprised of two segments, the polynucleotide recognitionsegment and one of the signalling components. The recognition segmentcomprises a polynucleotide probe that recognises the chosen targetpolynucleotide. The polynucleotide probe can undergo hybridisation witha complementary sequence of bases in the polynucleotide target to bedetected. The sequence of the probe polynucleotide should be at leastsix bases, preferably between six to fifty and optimally between twelveand thirty, in order to impart specificity to the probe and to ensuresecure binding between it and its target. However, such a base sequenceneed not be a single continuous complementary polynucleotide sequencebut can be comprised of two or more individual complementary sequencesinterrupted by non-complementary sequences. In addition, thecomplementary region of the probe can be flanked at the 3'- and5'-termini by non-complementary sequences, such as either thosecomprising the DNA or RNA vector into which the homologous sequence hasbeen inserted for propagation or a variety of blocking groups toprevent, for example, recognition in transcription processes. In eitherinstance, the probe as presented as an analytical reagent will exhibitdetectable hybridisation at one or more points with the samplepolynucleotides of interest.

Alternatively the polynucleotide recognition segment is comprised of twoor more polynucleotide sequences to be joined for example by ligation orcleaved, for example by restriction enzyme(s), after hybridisation tothe target. The target sequence of interest may be complementary to oneor more of the polynucleotide sequences.

It will be understood that the signalling components may be attached tothe above polynucleotide sequences in a number of ways. The componentsmay be attached via the same polynucleotide sequences or alternativelyvia adjacent polynucleotide sequences. Thus by way of example, thechelating ligand and the sensitising ligand may be attached to separatepolynucleotide sequences. The polynucleotide sequences may be contiguousor be separated by gaps to be "filled in" using appropriate dNTPs. Thelengths of the polynucleotide sequences will be determined by practicalconsideration. In general these will comprise at least eightnucleotides.

2.1. The polynucleotide segment

Methods for preparing the polynucleotide segment of the probe that issubstantially complementary to a target polynucleotide are well knownand routine in the art. One method involves recombinant DNA and anothercloning, as detailed in `M13 Cloning and Sequencing Handbook`, publishedby Amersham International (1983) and in `Molecular Cloning`, by T.Maniatis, E. F. Fritsch and J. Sambrook, published by Cold Spring HarborLaboratory (1982). Use of the polymerase chain reaction (PCR) can alsobe made to amplify target polynucleotide chains (K. Kleppe, et al. J.Mol. Biol., 1971, 56, 341-346; `PCR Technology`, ed by H. A. Erlich,Stockton Press, New York, 1989. Specific polynucleotides can also beprepared by using a DNA Synthesiser, such as the model 380B produced byApplied Biosystems, Foster City, Calif. or the Cyclone series ofsynthesisers supplied by Millipore Corporation, U.S.A. It will beunderstood that the term polynucleotide includes DNA, RNA and analoguesthereof which are able specifically to hybridise to the target.

The signalling component is a segment of the polynucleotide probe thatis involved in the energy transfer process leading to delayedluminescence. The signalling segment is attached to the polynucleotiderecognition segment either directly or through a linker arm. Thesignalling segment may be either the lanthanide chelator or thesensitising ligand.

The delayed luminescence should only occur when the polynucleotidesegment of the polynucleotide probe is hybridised with the targetpolynucleotide. The delayed luminescence should not occur in thepresence of hybrids if none of the hybrid strands is that of the targetpolynucleotide. The target polynucleotide, to which the polynucleotidesegment of the polynucleotide probe hybridises, must be one originatingfrom the sample. Thus, the polynucleotide probe must be presented to thetarget polynucleotide sample in single stranded form and should not havethe ability to form a doubly stranded hybrid with itself. If the lattersituation prevailed the doubly stranded probe polynucleotide willinteract with the intercalating agent such that the collaborativecombination of the sensitiser with the chelated lanthanide ion couldoccur, thus leading to the observation of delayed luminescence. Thiswill produce a false positive result. The formation of hairpin loops inthe polynucleotide probe can also result in the production of a falsepositive result in the presence of the intercalating agent. Selection ofthe probe sequence must take these possibilities into account but can beeasily checked for by carrying out the appropriate control measurements.The possible formation of doubly stranded material by the interaction ofthe probe polynucleotide with itself may be minimised by carefulselection of the base sequence to be searched for and by normally usingpolynucleotide probes not longer than about 30 base sequences.

2.2. Attachment of the signalling segment

The signalling segment of the polynucleotide probe may be either alanthanide chelating group, which binds the metal ion strongly andessentially irreversibly under the conditions used throughout the assayprotocol, or a sensitising ligand. This may be linked directly to thepolynucleotide segment or via a covalently bound linker arm. The pointof attachment to the polynucleotide may either be to a nucleic acid basegroup, a sugar group or a phosphate group. The base group may be eithera pyrimidine or purine unit. The attachment of the linker arm shouldpreferably be such that it does not interfere with the Watson-Crickpairing of complementary bases. Suitable positions are, for example,positions 5 and 6 of uracil, positions 5 and 6 or the exocyclic 4-aminogroup of cytosine, positions 7 and 8 of deazapurine, position 8 ofguanine and positions 8 and the exocyclic 6-amino group of adenine. Apreferred linker arm for attachment to the base moiety is allylamine seeEuropean Patent No. 063,879!.

Linkage through a hydroxyl group can be achieved, for example by anester or ether link to the 3'- or the 5'-terminal hydroxyl group ofdeoxyribose. Linkage to a phosphate group can be by means of an alkylphosphate ester link, either to the 3'- or the 5'-terminal positions ofthe polynucleotide. The linker arm should be chosen so that it is stableunder the conditions used in the assay protocol. The method forattaching the linker arm should be any that does not result in themodification or blocking of the functional groups of the bases requiredfor hybridisation or the cleavage of the base from the sugar.Conveniently the 5' and/or 3' termini, preferably the 5' terminus, areused for attachment.

2.3. The linker arm

The linker arm comprises a group of atoms joining the polynucleotiderecognition segment to the chelator-metal complex or the sensitiser. Thelinker arm can be joined to the polynucleotide recognition segment byany number of methods. The linker arm must have a first functional groupby means of which it can be attached to the recognition segment and asecond functional group by means of which it can be attached to eitherthe lanthanide metal chelator or the sensitising ligand. The linker armcan be attached for example by means of a carbon-carbon single bond,carbon-carbon double bond, carbon-carbon triple bond, carbon-nitrogensingle bond, carbon-nitrogen double bond, carbon-oxygen single bond,carbon-sulphur single bond, carbon-silicon single bond, sulphur-nitrogenbond, sulphur-oxygen bond, phosphorous-oxygen bond, orphosphorous-nitrogen bond.

Suitable functional groups include but are not limited to, hydroxylgroups, amino groups, thio groups, alkyl sulphates, and halides. It isnot necessary that the linker arm be attached to the polynucleotiderecognition segment as one fragment.

2.4. Attachment of the linker to the polynucleotide recognition segment

Where the linker is attached to the 5' or 3' terminus of thepolynucleotide, convenient linkages include phosphate, carboxy or etherlinkages, particularly phosphate linkages. Suitable phosphate linkersinclude aminoalkylphosphoryl groups, especially those comprising a C1-12alkyl chain, especially a C6 alkyl chain. These linkers may be readilyattached to synthetic oligonucleotides during solid-phase synthesis, seefor example S. Agrawal et al, Nucleic Acids Research, 1986, 14, 6227 andWO-88/02004 (Applied Biosystems). The amino group may then be used forthe attachment of the chelator-metal complex or the sensitizer.Alternatively the linker can be constructed by attaching a firstfragment to the recognition segment, followed by the attachment of asecond fragment to the first fragment.

Examples of suitable first fragments include:

--NH--CH₂ --CH═CH--, SH--CH₂ --CH₂ --CH═CH--, --NH--CH₂ --CH₂ --O--CH₂--CH═CH--, --(CH2)_(n) --O-- where n is an integer from 1 to 20.

Examples of suitable second fragments include those introduced by meansof:

    ______________________________________                                        N--O--CO--R; R--C(═NH)--OR;                                                                          R--CO--O--CO--R;                                   N-hydroxysuccinimide                                                                       imidates      anhydrides                                         esters                                                                        R--N═C═S;                                                                          R--CO--SR  and                                                                              R--(C═S)--SR                                   isothiocyanates                                                                            thioestets    dithioesters                                       ______________________________________                                    

Second fragments or the chelator or the sensitizer may be introduced viathe amine reactive functions, for example:

R--COO--Su (wherein Su represents a succinimidyl group), R--(C═NH)--OR,R--COO--CO--R, R--N═C═S and R--CS--SR or by thiol reactive functions--R--O--C(═O)--CH₂ --X (wherein X=a halide group), R--Ma (wherein Marepresents a maleimido group), R--S--S--Y (wherein Y is preferably anelectron withdrawing group such as a pyridyl group). In all the abovefragments R represents a linker group as hereinbefore defined.

Other general methods for attaching a linker arm onto a polynucleotidebase are discussed in J. L. Ruth and D. E. Bergstrom, J. Org. Chem.,1978, 43, 2870; D. E. Bergstrom and M. K. Ogawa, J. Amer. Chem. Soc.,1978, 10, 8106; and C. F. Bigge, P. Kalaritis, J. R. Deck and M. P.Mertes, J. Amer. Chem. Soc., 1980, 102, 2033. One preferred method isthe one disclosed in detail in European Patent Application No. 063,879,which is hereby incorporated by reference. The method involves reactinga linker arm or a linker arm fragment containing an alpha-vinyl groupwith a mercurated base in the presence of K₂ PdCl₄, wherein the mercuryis bound as Hg+ to the position of the base which is to react with thelinker arm.

There are no particular size or content limitations for the linker armprovided that it can fulfil its stated purpose. The linker arm cancontain from about two carbons to any number of carbons. The linker armcan contain heteroatoms and unsaturations, The linker arm can comprisealiphatic, alicyclic, aromatic or heterocyclic groups. It convenientlycomprises --(CH₂)_(n) --. It may however include other groups such as--O--, --CHOH--, --COO--, and --CH₂ CH₂ --O-- which help maintain watersolubility. When the linker is --(CH₂)_(n) -- and n is eight or morethen it should preferably not comprise methylenes alone but includeother groups as described above, or be introduced via two fragments asdescribed above.

Attachment of the linker arm to the sugar group of a polynucleotide canbe by means of a Schiff base to the 1'-aldehyde following depurinationor depyrimidation of preselected bases or it can be to the 2'-hydroxy inthe case when the sugar is ribose. Attachment of a linker arm to thephosphate moiety can be by alkylation of the phosphate group, see U.S.Pat. No. 4,469,863, which is hereby incorporated by reference.

When the linker arm is attached to the base group, it is preferable toattach it to the base before formation of the polynucleotide. This isbecause the reaction conditions that may be required to attach thelinker arm to the base may cause undesirable side reactions to apolynucleotide. Furthermore, attachment at the polynucleotide level maygive inconsistent and irreproducible yields. Attachment at thenucleoside or nucleotide level permits the modified nucleoside ornucleotide to first be purified and then to be incorporated into apolynucleotide. The incorporation can either be by cloning, for example,in an M13 vector, or by synthesis in a polynucleotide synthesiserinstrument as described above.

For incorporation by a M13 vector, the modified nucleotide must be arelatively efficient substrate for the commonly studied nucleic acidpolymerases. Thus, the linker arm should not sterically interfere eitherwith the active site on the enzyme or with the complementary basepairing of the modified nucleotide. Substitution at positions that alterthe normal `anti` nucleoside conformation should also be avoided sincesuch conformational changes usually render the modified nucleotide apoor substrate for the polymerase enzymes.

When the linker arm is attached to the 1'-aldehyde of the sugar, thelinker arm must be attached following the formation of thepolynucleotide portion of the polynucleotide probe. This is becauseattachment of the sugar requires a free aldehyde at the 1'-position ofthe sugar. The free aldehyde is formed by depurination ordepyrimidation. A group comprising a sugar and a phosphate without abase is not a substrate for the polymerase enzymes. Thus the linker armmust be attached by first selectively depurinating or depyrimidinatingthe desired polynucleotide sequence and then attaching the linker arm tothe sugar by means of the aldehyde. When the linker arm is attached tothe 2'-hydroxy group of a ribose sugar, the linker arm can be attachedat the nucleoside, nucleotide or polynucleotide level. This is becausenucleotides modified by a linker arm can be incorporated into apolynucleotide by means of a polynucleotide synthesiser instrument. Whenthe linker arm is attached to the phosphate, the linker arm ispreferably attached at the nucleoside or nucleotide level so that theattachment is not at positions other than at a phosphate.Phosphoramidite technology may be used in a nucleic acid synthesiser toincorporate the linker to the 5' or 3' end of the polynucleotide.

2.5. Attachment of the Chelator to the Linker

A chelator is a group which can sequester and bind a metallic cation.The chelator has two or more functional groups which interactnon-covalently with the metal. The attachment of metal chelating groupsto polynucleotides is known in the art. See European Patent Nos.097,373, 150,844 and 157,788, which are hereby incorporated byreference.! The chelator acts to shield the lanthanide from water. It isa key requirement in this invention that the chelator does not saturateall the possible binding sites around the lanthanide ion since spacemust become available around the ion to accommodate the sensitisingligand.

Examples of chelators, not meant for limitation, areethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA); trans-1,2-diaminocyclohexanetetraacetic acid (DCTA);1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diacetic acid andpolycyclic aza-crown systems such as bicyclo5.5.2!1,4,7,10-tetraazacyclotetradecane-4,10-diacetic acid. Modifiedderivatives of the above, such as 1-phenylethylenediaminetetraaceticacid, allow for sites where linking groups can be placed in the aromaticring, such as the diazonium or isothiocyanate derivatives.

Other examples of suitable chelators include polyfunctional compoundssuch as crown ethers, caged compounds, clathrates with suitable donorgroups to bind the lanthanide. These groups will conveniently includenitrogens, oxygens, for example on ethers or carboxylic acid groups. Theselection of a suitable chelators will be apparent to the chemist orordinary skill. Other chelators and the factors influencing theireffectiveness are reviewed in `Cation Binding by Macrocycles` ed. Y.Inoue and G. W. Gokel, pp 203-251 by T. M. Fyles published by MarcelDekker, New York 1990. A further review is provided by Arnaud-Neu inChemical Society Reviews, 1994, 23, 4, 235-241; this describes interalia the use of coronands, cryptands, calixarenes and modified versionsthereof to form stable lanthanide complexes where not all availablesites on the lanthanide ion are occupied.

The chelator can be attached to its linker by a number of groups.Examples, not intended for limitation, are: --O--, --NH.CO--,--NH.C(NH)--, --NH.C(S)--, --N═N--, --NH.SO2--, --S--, --O.PO2--,--O.SO2--, --NH--N═N--, ----NH--CH2--, --CH2.NH--, --NR--, --O.CH2--,--O.CO--, --NH.CO.CH2.S--, --NH.CO.CH2.NH--, --O.CH2.CH2.O--,--O.CO.CH2--, --S.CH2--, and --O.CO.NH--.

Alternatively one of the carboxyl groups of a chelator as outlined abovemay be used for attachment of the linker. The use of anhydrides, forexample EDTA anhydride, is particularly convenient. It will beappreciated that in certain circumstances the chelator may containgroups which act as sensitisers for the metal.

Varying conditions can be used for attaching a chelator to a linker arm.Generally any pH range from about 4 to about 10, preferably from about 5to about 8, any temperature from about 20° C. to about 100° C., anysolvent, preferably water, and any buffer or catalyst can be used aslong as the pH, temperature, solvent or buffer does not modify any ofthe groups or moieties of the polynucleotide. Thus, for example,reagents or conditions that can depurinate or deaminate thepolynucleotide should be avoided. There are also relatively fewlimitations as to reaction times. The optimum pH, temperature, solventor reaction time for attaching the chelator to a linker arm will dependon the linker arm, the chelator, and the functionalities to be reactedand may be determined by the scientist of ordinary skill.

The stoichiometries of the reactants required for these linkingreactions vary widely. Generally, an excess of the component that ismore easily prepared will be used for the attachment of the chelator tothe polynucleotide. Again, the amounts will vary depending upon thereaction conditions, the chelator, the linker arm and their reactingfunctional groups.

The chelator can be attached to the linker arm either afterincorporation of the linker arm to the polynucleotide or beforeincorporation of the linker arm into the nucleotide. The only limitationis that the chelator cannot be attached before incorporation if itinterferes with polynucleotide synthesis. The binding entity cancomprise one chelator or more than one chelator. For the polynucleotiderecognition segment the chelator can be attached at terminal positionsor at non-terminal positions of the polynucleotide probe. The greaterthe number of chelators the more sensitive the binding entity will be.However, the chelators should not be present in such numbers thateffective complexing of the analyte to the binding entity issubstantially prevented. The number of chelators that can be attachedwill depend on the composition, the size and the length of therecognition segment.

2.6. Attachment of the metal

Of the lanthanide metal chelates those of terbium, europium, samariumand dysposium can exhibit long-lived luminescence of up to themillisecond range. Terbium emits in the range 480 to 630 nm and europiumin the range 580 to 700 nm. Europium is the preferred metal. Neither ofthese ions show strong absorbance (extinction coefficients) in aqueoussolution and chelates with non-sensitising ligands, for example EDTA,also show very low extinction coefficients, the weak absorptionoccurring in discrete regions, of 270-320 nm and about 488 nm forterbium and of 320-360 nm and about 580 nm for europium. The excitedstate of these metals can be reached by use of energy transfer fromsuitable sensitisers. Singlet energy sensitisers, employing throughspace Forster energy exchange mechanisms, are inefficient since it isbelieved that the excited state of these lanthanide ions are formallyforbidden and the processes require triplet state sensitisation. Tripletstate sensitisers do not efficiently transfer energy through space,requiring close contact, such as collisions in order to transfer theirenergy. The excitation process can be achieved efficiently by use oftriplet sensitisers which can act as ligands, i.e. they are always heldin close contact with the metal ion. The metal ion can be chelated tothe chelator by stirring a solution of the latter in a solvent with asolution of the metal salt, such as a halide or nitrate salt in therange of pH 5 to 10, preferably between 6 and 8. The formation of thechelated lanthanide may be slow and can take several hours. However,once formed because the binding constants are high, dissociation isextremely slow and the process is essentially irreversible within therange of pH 5 to 10.

As an alternative to the attachment of the chelator to the linker onecan attach the sensitiser, as detailed below.

3. Description of the Sensitiser

The sensitiser is the antenna for the energy transfer process. It actsto efficiently pass on its excited state energy to the lanthanide whenthey are in close proximity, as accomplished by ligation. The sensitisercan be any of a range of possible organic, aromatic or heteroaromaticsystems that can act as triplet sensitisers. A key requirement in thisinvention is that the systems can act as ligands for the lanthanide ionwithout displacing the chelating group around the ion; both thesensitiser and the chelator must be able to reach and bind to thelanthanide ion at the same time and when in the presence of the analyte.Suitable ligand sensitisers include but are not limited tobeta-diketones of the general formula I, where R1, R2, R3=alkyl,heterocyclic, aromatic or heteroaromatic groups, ethers, esters, amides,etc.

Heterocyclic systems include but are not limited to substituteddipyridyl compounds of the general formula II and phenanthrolines of thegeneral formula III where R1, R2=a chelating group, such as --CO2H,--CO.NHR, --CO.NR2, --CO.NR1,R2, --CO.NH.OH, --CH2CO2H, --CH2.CO.NHOH,--CH2PO.(OH)2, --CH2.PO(R)OH, --CH2OH, --CH2SH, --PO(OH)2, --PO(R)OH,--CH2N(CH2.CO2H)2. Other convenient heterocyclic systems have beendescribed in the literature and will be apparent to the scientist ofordinary skill.

The sensitiser may also bear a linker (R3); the linker arm comprises thegroup of atoms joining the sensitiser to either the probe polynucleotideor the intercalator. The linker can be attached to the sensitiser by anumber of groups. Examples of such groups, not intended for limitationare those detailed for the attachment of the linker to the chelator.

4. Description of the duplex binder

A number of aromatic agents or dyes are able to bind to double-strandedpolynucleotide either by the process of intercalation, in which theagent inserts itself between adjacent sets of the hydrogen-bonded basepairs or by binding in either the major or minor grooves of the duplex.The double strand polynucleotide can be DNA--DNA, DNA--RNA or RNA--RNA.

A result of intercalation is the spreading of adjacent base pairs toabout twice their normal separation distance, leading to an increase inmolecular length of the duplex. Some unwinding of the double helix mustalso occur in order to accommodate the intercalator see M. J. Waring andL. P. G. Wakelin, Nature, (London), 1974, 252, 653; L. P. G. Wakelin,Med. Chem. Rev., 1986, 6, 275.! Examples of intercalating agents, notintended for limitation, are acridine dyes, e.g. acridine orange andacriflavine, the phenanthridines, e.g. ethidium, anthracyclines, e.g.adriamycin, quinoxalones, e.g. dactinomycin, the phenazines, quinolines,anthracenes, furocoumarins, and phenothiazines.

The intercalators form reversible complexes with the duplex DNA, therates of association and dissociation being fairly rapid and temperaturedependent; the association constants being in the range of 10⁴ -10⁸mol⁻¹, usually in the order of 10⁶ mol⁻¹. It is important that the rateof this process is fairly fast so that intercalation between differentbase pairs occurs until the agent is within reaching distance of thechelated lanthanide ion in order to form the active complex.

Example of groove binding agents, not intended for limitation, are thepolypyrrole antibiotics netropsin and distamycin, the diamidines beveniland hydroxystibamidine, Hoechst 33258, DAPI(4',6-diamidino-2-phenylindole), chromomycin, olivomycin, mithramycinand crystal violet.

The duplex binder is preferably attached to a linker; a linker armcomprises an atom or group of atoms joining the intercalator to eitherthe sensitising ligand or the chelator. Examples of such groups but notintended for limitation are those detailed for the attachment of thelinker to the chelator.

5. The Linked Duplex binder--Sensitiser

The intercalator linked sensitiser compounds described herein under thegeneral formula (IV), where the component parts are described insections 3 and 4 above, are new compounds and are claimed as part ofthis invention. Linkage of the duplex binder to the sensitizer/chelatoris conveniently effected using a linker arm which will convenientlycomprise --(CH2)_(n) -- where n is between 2 and 20, preferably between4 and 12. The linkage to the sensitiser or chelator and to the duplexbinder will depend on the chemical nature of those moieties and anyconvenient means that preserves the function of those moieties may beemployed. Such linkages will be apparent to a chemist of ordinary skill.Preferably the linkage will not contain bulky atom groupings. Preferablythe linker in the vicinity of the duplex binder will not be hydrophilicand preferably will not contain negative charges.

6. The Analyte

The method of the invention can be used to detect a targetpolynucleotide, for example, from any convenient eukaryotic orprokaryotic species such as a microorganism, a plant cell or a mammaliancell. The microorganism can be a bacteria, fungus, virus or yeast. Thetarget polynucleotide can be one that is unique for a particularpathogenic virus, one that is present in a mutated mammalian gene thatresults in the production of a non-naturally acting protein, or one thatimparts resistance to a bacteria. It can also be a productpolynucleotide arising from the amplification of a polynucleotide by useof the polymerase chain reaction, or as prepared by cloning methods. Aparticular product polynucleotide arises from the use of the polymerasechain reaction (PCR) as decribed for example in European Patent No. 0201 184 or from the use of the Amplification Refractory Mutation System(ARMS) as claimed in European Patent No. 0 332 435 B1 (Zeneca Limited).Further product polynucleotides may be obtained by the use of Q-betareplicase as described in PCT patent application, publication no.WO-87/06270; by the use of the transcription-based nucleic acidamplification (TAS) of Siska Corporation as described in PCT patentapplication, publication no. WO-88/10315; by the use of single primeramplification (SPA) as described in European patent application,publication no. 0 469 755 (SYNTEX); by the use of sustained sequencereplication (3SR); by the use of ligase chain reaction (LCR); or by theuse of repair chain reaction (RCE).

PCR amplification and, for some applications ARMS amplification, is apreferred first step in the detection methods of the present invention.

The test sample to be assayed can be any medium of interest, and willusually be a liquid sample of medical, veterinary, environmental,nutritional, or industrial significance. Human and animal specimens andbody fluids can be assayed by the present method, including urine, blood(serum or plasma), amniotic fluid, milk, cerebrospinal fluid, sputum,fecal matter, lung aspirates, throat swabs, genital swabs and exudates,rectal swabs, and nasopharyngeal aspirates. Where the test sampleobtained from the patient or other source to be tested containsprincipally double stranded nucleic acids, such as contained in cells,the sample will be treated to denature the nucleic acids, and ifnecessary first release nucleic acids from cells. Denaturation of thenucleic acids is preferably accomplished by heating in boiling water oralkali treatment, e.g. 0.1M sodium hydroxide, which, if desired, can besimultaneously used to lyse cells. Also, release of nucleic acids can,for example, be obtained by mechanical disruption, (freeze/thaw,abrasion, sonication) physical/chemical disruption (detergents such asTriton, Tween, sodium dodecylate, alkali treatment, osmotic shock, orheat) or enzymic lysis (lysozyme, proteinase, pepsin). The resultingtest medium will contain the nucleic acids in single stranded form whichcan then be assayed according to the present hybridisation method.

FIG. 1 illustrates the key parts of the invention as exemplified by useof an intercalator. It is a requirement that the links used do notinterfere with the possibility of the sensitiser and chelator reachingeach other. They should be selected such that their overall length isneither too long nor too short. Typically the number of atoms in eachlink should be between two to twenty atoms such that the combined lengthof the two links is between four and fourty atoms, optimally between tenand thirty atoms.

The test sample is conveniently obtained from target eukaryotic cellsand comprises samples of genomic DNA and/or RNA for the analysis of, forexample, inherited or acquired disease, the determination of identityincluding paternity, predisposition to disease or a particularcondition; and population polymorphisms.

The invention is of particular use in HLA typing and the detectionand/or diagnosis of cystic fibrosis, sickle cell anaemia and cancer.

7. Multiplex Testing

It will be appreciated that the methods of the invention may be used todetect more than one target polynucleotide simultaneously.

A convenient method comprises the use of one or more Forster energytransfer acceptor able to accept energy from the luminescent lanthanidecomplex of the invention. A number of different such acceptors thatwould emit light of a different wavelength are selected and used suchthat each energy transfer event was indicative of a different nucleicacid sequence. Luminescence energy transfer has been disclosed for thedetection of DNA, see for example P. R. Selvin et al. J. Am. Chem. Soc.116, 6029-6030 (1994). A number of variations using energy transferacceptors are possible:

Variation 1: The energy transfer acceptor is coupled to thepolynucleotide probe of the invention. Thus for multiple detection, eacholigonucleotide probe, of varying sequence, emits light of a distinctwavelength which is indicative of the presence of the target nucleicacid complementary to that probe. Coupling of the energy transferacceptor to the probe is at any convenient point that does notinterefere with its function. Preferably it is positioned within the Rovalue for the energy transfer pair selected. Conveniently, where thesensitisor or chelating ligand is positioned at one terminus of thepolynucleotide, the energy transfer acceptor is attached to the oppositeterminus. Alternatively it is attached to a base.

Variation 2: The energy transfer acceptor is coupled to a polynucleotideprobe that has been selected to bind to the target nucleic acid adjacentto the polynucleotide bearing the sensitiser or chelating ligand. Asignal is thus observed only when both polynucleotide probes are boundto the target nucleic acid. Again the attachment to the polynucleotideprobe is at any convenient point that does not effect function and,together with the selection of the polynucleotide probe sequence, isselected such that a useful degree of energy transfer from theluminescent lanthanide complex takes place.

Variation 3. Variation 2 is employed but the adjacent polynucleotideprobe bearing the energy transfer acceptor is an allele-specific probe.Thus the presence or absence of acceptor emission determines thepresence or absence of a particular allele on the target DNA. A furtheruse of this is in conjunction with a mismatched polynucleotide for thedetection of phase of closely linked mutations, for example in the HLAlocus. In this manner the wavelength of light emission will indicate thepresence of one or both mutations.

The use of energy transfer acceptors is believed to have certainadvantages which also apply to the detection of single nucleic acidsequences (non-multiplexed assays). For example the use of adjacentpolynucleotide probes as herein described gives an extra level ofspecificity to the assay which may be beneficial in certain situations.Furthermore the overall efficiency of light emission for systemsemploying luminescence energy transfer is believed to be higher thanthat involving lanthanide emission alone and hence a more sensitiveassay will result.

Convenient energy transfer acceptors include certain fluorescentproteins (for example allophycocvanin), certain phthalocyanines, Cy-5(Biological Detection Systems) and other fluorescent compounds which maybe excited by lanthanide emissions. Suitable compounds and means forattaching them to the assay components may be identified using methodsand materials outlined above and/or routine experimentation. Aconvenient appreciation of the issues involved in selection of suitableacceptors, and of the distances between donor and acceptor, is providedby Selvin op cit.

In a further aspect an energy transfer acceptor conjugated to a duplexbinder. This novel entity will bind to the duplex DNA in the vicinity ofthe luminescent lanthanide complex, accept the energy from thelanthanide emission and emit light. The efficiency of light emission forsystems employing luminescence energy transfer is believed to be higherthan that involving lanthanide emission alone and hence a more sensitiveassay will result.

Alternatively multiplex testing is conveniently effected using differentemission spectra i.e. using excitation wavelengths and/or differentlanthanides to distinguish between the hybridisation products atdifferent target polynucleotides. Examples of suitable systems will beapparent to the scientist of ordinary skill.

Whilst we do not wish to be bound by theoretical considerations thenumber of target polynucleotides which may be detected simultaneously islimited principally by practical considerations.

8. Further Features

A further feature of the invention is the use of one or more controlreactions to ensure the validity of the results obtained. A particularcontrol reaction is provided by the use of a control polynucleotideproducing upon hybridisation a different emission spectrum to bedetected.

In a still further aspect of the invention measurement of the rate ofsignal change during hydridisition may be used for diagnostic purposes.By way of non-limiting example the rate of signal change may beindicative of target polynucleotide copy number. This could for exampleindicate whether an individual is homozygous or heterozygous for aparticular allele of a genetic locus. Alternatively it could indicatethe presence of a mutant allele against a background of normal alleles.This may be of particular interest in the detection and diagnosis ofcancer. The rate of signal change may also reflect the composition ofthe hybridisation probe and its target polynucleotide and is a usefulanalytical tool. As previously mentioned the methods of the inventionare of particular use in conjunction with the Amplification RefractoryMutation Systems (ARMS). One or more ARMS amplification primers may beprovided with signalling components for use in the invention.Alternatively the products of ARMS amplification are used as the targetpolynucleotide for analysis.

Convenient assay formats include multiplex reactions. If information isrequired regarding individual mutations then separate sample aliquotsare analysed for example on different microtitre plates. Alternatively,in a preferred aspect of the invention, analytes are held and movedwithin a closed system such as provided by the "pouch" technologydeveloped by Eastman Kodak. This may reduce contamination and aid theinterpretation of results.

9. Assay Kits

The invention also relates to assay kits for the detection of analytessuch as target polynucleotides. Such kits conveniently comprise morethan one of the following features i.e. one or more (i) polynucleotideprobe(s), (ii) sensitiser(s), (iii) intercalator(s), (iv) linkedintercalator--sensitiser(s), (v) buffers, (vi) PCR amplification primersand (v) instructions for use. Some or all of the assay components may bepresent at the start of any amplification procedure, for example theprimers and/or probe(s) and/or intercalator and/or (linked) sensitiser.

The above kits are conveniently adapted for use in the methods of theinvention. By way of non-limiting example the kits may comprise a"pouch" system or single reaction vessel and/or microtitre plates. Theabove species preferably comprise light transparent vessel(s) tofacilitate signal detection.

10. Sequence-dependent intercalation

It is clear from the foregoing that the second component of the energytransfer system is localised, by intercalation, within a specific,proximate distance of the first component. It will be appreciated thatthe nucleotide sequence of the polynucleotide probe may be adapted sothat a signal is only produced when probe binding is correct. That is tosay one or more potential regions of non-complementarity may beincorporated into the polynucleotide probe sequence so that whendiagnostic mismatches are present upon hybridisation with the targetpolynucleotide intercalation cannot occur and no signal is produced. Ifdesired additional destabilising mismatches may be employed to increasethe sensitivity of this embodiment.

11. Instrumentation

The methods of the invention may employ a variety of instrumentation formeasuring fluorescence, depending on the format of the assay. For assaysperformed in microtitre plates fluorimeters capable of measuringtime-resolved fluorescence in plates are preferred, for example theDELFIA fluorimeter (Wallac). Alternatively, suitable fluorimeters forcuvettes and tubes are available. For measuring PCR and ARMS productsinstrumentation may be devised for measuring the signal in theamplification vessel without having to open the vessel (and optionallyduring the course of the amplification reaction). This avoidscontamination problems.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be illustrated but not limited by reference tothe following Figures and Examples wherein:

FIG. 1 illustrates binding of probe DNA to target DNA. The intercalator,link, sensitizer, lanthanide ion, chelator and the link between chelatorand probe DNA are shown.

FIG. 2 shows the output of the assayed solution described in Example 1at 112× dilution and with a concentration of probe and targets atapproximately 1.7×10⁻ 8M. Curve a is with matching target A and curve bis with non-matching target B.

FIG. 3 shows the output of the assayed solution described in Example 4with normal Target 3860 and normal Probe 3922, both at 2×10⁻⁷ Mconcentration.

FIG. 4 shows the output of the assayed solution described in Example 4with mutant Target 3288 and normal Probe 3922, both at 2×10⁻⁷ Mconcentration.

FIG. 5 shows the output of the assayed solution described in Example 4with normal Probe 3922 at 2×10⁻⁷ M concentration. No target was present.

FIG. 6 shows the output of the assayed solution described in Example 4at 64× dilution (i) with normal Probe 3922 and mutant Target 3288, bothat 3.1×10⁻⁹ M concentration solid line! and (ii) with normal Probe 3922and normal Target 3860, both at 3.1×10⁻⁹ M concentration dotted line!.

In FIGS. 2-6 above intensity is shown on the Y axis and wavelength innanometres on the X axis.!

METHODS AND MATERIALS

Preparation of the Probe nucleotide (1):

This was achieved using an Applied Biosystems polynucleotide synthesiserusing the phoshoramidite method. A 6-aminohexyl group was introducedonto the 5'-terminal phosphate group, to give structure (1) by use of anextra cycle of phosphoramidite synthesis with9-fluorenylmethoxycarbonylaminohexyl beta-cyanoethylN,N'-diisopropyl-aminophosphite in the coupling reaction.

Preparation of Chelator (2):

The oligonucleotide (1) (250 μl, 10⁻⁶ M), was adjusted to pH 7.5 byfirst adding 100 ul 10 mM Na₂ CO₃ and then adding dilute HCl (0.1M). Thesolution was stirred with EDTA bis anhydride (25×) at room temperaturefor 5 h. To the stirred solution was then added EuCl₃.6H2O (25×) andstirring continued for another 5 h. The mixture was centrifuged and thesupernatant liquid was passed through a Sephadex column (NAP 5 column,Pharmacia), using a 10 mmol Tris buffer, adjusted to pH7.5 with 0.1M HCl, as eluant and collecting 50 μl fractions. Fractions were monitored byabsorbance at 260 nm; those fractions showing absorbance were combinedand stored at 2° C. until required.

Preparation of the Intercalator (3):

a. 5-Nitro-2,9-dimethylphenanthroline (4) Neocuproine(2,9-dimethylphenanthroline hemihydrate; 10 g) was added to cold fumingsulphuric acid (44 ml) in portions with stirring at room temperature andthen fuming nitric acid (50 ml) added before heating the solution at140° C. under nitrogen for 1 h. After this time the reaction mixture wascooled and poured cautiously onto crushed ice, before neutralising withsolid sodium carbonate to pH6. The yellow solid produced was collectedand purified by reprecipitation from 3M sulphuric acid solution to givethe nitrated product (4) (6.6 g, 50%), m.p. 176°-180° C. (dec.). Thematerial analysed as the hemihydrate; Calcd. for C14H11N302.1/2H2O: C,64.12; H, 4.60; N, 16.02. Found: C, 64.52; H, 4.23; N, 15.68%.

b. 5-Nitro-2,9-bis-(trichloromethyl)-1,10-phenanthroline (5) The nitrocompound (4) (5.06 g, 20 mmol) in carbon tetrachloride (150 ml) andchloroform (25 ml) was heated at reflux in the presence ofN-chlorosuccinimide (18.7 g, 140 mmol) and a catalytic amount of3-chloroperbenzoic acid. After 24 h the mixture was cooled to roomtemperature and filtered. The filtrate was washed several times with 10%w/v sodium carbonate solution, dried and the solvent removed to give thecrude product; a further crop was obtained from the filtered solids bytrituration with chloroform and treating in a similar fashion, to give atotal yield of 7.08 g (77%) Material (5) was obtained analytically pureby column chromatography through silica gel using chloroform as eluant.The pure material showed m.p. 228°-231° C. Found: C, 36.16; H, 1.11; N,8.91. C14H5N302Cl6 requires C, 36.56; H, 1.10; N, 9.14%.

c. 5-Nitro-1,10-phenanthroline-2,9-dicarboxylic acid (6)

The hexachloride (5) (1.5 g, 3.3 mmol) was mixed with 98% sulphuric acid(8 ml) and heated to between 80°-90° C. under nitrogen. After 6 h theviscous solution was poured over crushed ice, precipitating the diacidas a pale yellow solid. The solid was collected and recrystallised fromhot aqueous tetrahydrofuran to give the pure diacid (0.9 g, 89%); m.p.218°-220° C., Found: C, 52.46; H, 2.77; N, 12.95. C₁₄ H₇ N₃ O₆.1/2H2Orequires C, 52.18; H, 2.50; N, 13.04%.

d. 5-Nitro-2,9-bis(methoxycarbonyl)-1,10-phenanthroline (7)

The hexachloride (5) (2.0 g, 4.35 mmol) was mixed with 98% sulphuricacid (5 ml) and heated to 90° C. under nitrogen for 2 h. The mixture wascooled in ice and then added slowly to methanol (10 ml) After heating atreflux for a further 45 min, the excess of methanol was removed in vacuoand the residue neutralised to pH 6-7 with saturated aqueous sodiumcarbonate solution. The crude product was collected by vacuum filtrationand dried to give the pure diester (1.27 g, 86%); m.p. ca. 260° C.(dec.). Found: C, 56.42; H, 3.24; N, 12.05; C₁₆ H₁₁ N₃ O₆ requires C,56.31; H, 3.24; N, 12.31%.

e. 5-Amino-2,9-bis(methoxycarbonyl)-1,10-phenanthroline (8) The nitrocompound (7) (1.0 g, 3 mmol) in methanol (100 ml) was heated withcyclohexene (1.4 g, 17 mmol) and Pd/C10%, 0.2 g) and reflux continuedfor 3 h. The mixture was cooled and filtered through Celite, washing theresidues with more methanol (50 ml) until no more colour exuded. Thecombined filtrates were evaporated to afford the product amine as abright yellow solid (0.73 g, 77%); m.p. ca. 240° C. (dec.) M/e 311 (M+;57%), 253 (86), 195(56).

f. 5-Amino-1,10-phenanthroline-2,9-dicarboxylic Acid (9) Thenitro-diacid (6) (0.3 g, 0.93 mmol) in formic acid (15 ml) was treatedwith Pd/C (10%, 0.3 g) and the mixture heated to reflux for 2 days undernitrogen. The mixture was cooled, filtered and the solids washed withmore formic acid (10 ml) before collecting the filtrate and evaporatingoff the solvent formic acid to yield the product amine as an orangesolid (0.26 g, 96%). This material was extremely insoluble in mostorganic solvents. It was characterised by conversion to thecorresponding dimethyl ester (8). Thus a small sample (30 mg) of theacid (9) was stirred in methanol (3 ml) containing one drop of 98%sulphuric acid at room temperature for 16 h. The solution wasneutralised with solid sodium hydrogen carbonate, filtered and thesolvent removed to yield the diester (8), identical in its physical andchromatographic behaviour to the material described above.

g. 5-(6-Bromohexanoyl)amino-2,9-bis(methoxycarbonyl)-1,10-phenanthroline(10) The amine (8) (0.5 g, 1.6 mmol) was reacted with 6-bromohexanoylchloride (0.425 g, 2 mmol) in dry chloroform (5 ml) containing an excessof Hunig's base. After stirring at room temperature for 2 h the solutionwas washed with water and dilute HCl and, finally, water before drying ,filtering and evaporation of the solvent to afford the amide, (0.65 g,85%), m.p. 129°-132° C. dec.); Found: C, 52.18; H, 4.66; N, 8.24; Br,15.91. C₂₂ H₂₂ N₃ O₅ Br.H₂ O requires C, 52.18; H, 4.77; N, 8.30; Br,15.78%.

h. 5-6-(N-Phenanthridinium)hexanoyl!amino-2,9-bis(methoxycarbonyl)-1,10-phenanthrolineBromide (11) Phenanthridine (0.52 g, 2.9 mmol) was heated to 120° C.under nitrogen to its melting point and then, to the melt was added,portionwise over 10 minutes, the bromide (10) (0.525 g, 1.04 mmol). Themixture was heated at 120° C. for 90 min before cooling and dissolvingthe solid product in chloroform (8ml). To the solution was added ether(15 ml) to form a yellow precipitate, which was collected by filtrationto give the salt (11) (0.677 g, 95%). The product was recrystallisedfrom water to give pale yellow needles of the monohydrate, m.p. 160° C.(dec.). Found: C, 61.45; H, 4.66; N, 8.23. C₃₅ H₃₁ N₄ O₅ Br.H₂ Orequires C, 61.32; H, 4.85; N, 8.17%.

i. 5-6-(N-phenanthridinium)hexanoyl!amino-1,10-phenanthroline-2,9-dicarboxylicAcid Bromide (3) The dimethyl ester (11) (0.10 g, 0.15 mmol) was addedto distilled water (7 ml), the pH adjusted to 4 with dilute HBr and themixture heated to reflux, whereupon the solid slowly dissolved toproduce a yellow solution. After 20 h the solution was filtered toremove traces of solid, and then freeze dried to give the required acid(50 mg, 52%), m.p. >180° C. (dec.). The material was characterised byre-esterification with methanol under HBr acid catalysed conditions toreform the ester (11).

Preparation of the intercalator (12)

j. 4-(Nitrophenyl)-2,9-dimethyl-1,10-phenanthroline (13)4-Phenyl-2,9-dimethyl-1,10-phenanthroline (0.5 g, 1.75 mmol) in 98%sulphuric acid (4 ml) was cooled to <10° C. in an ice-salt bath beforeadding, dropwise, a mixture of 98% sulphuric acid (1 ml) and 65% nitricacid (1 ml) keeping the temperature below 15° C. After addition themixture was allowed to warm to ambient over 20 min before pouring ontocrushed ice and then neutralising with 7N sodium hydroxide to pH 7, toproduce a beige precipitate. The precipitate was extracted intochloroform (3×25 ml) washed with brine (25 ml), dried, filtered andevaporated to give the product as a mixture of ortho-, meta- andpara-nitrophenyl isomers (0.58 g, 100%).

k. 4-(Nitrophenyl)-2,9-bis(trichloromethyl)-1,10-phenanthroline (14) Thenitro-compound (13) (0.5 g, 1.6 mmol) was dissolved in chloroform (5 ml)and then diluted with carbon tetrachloride (30 ml). To the solution wasadded N-chlorosuccinimide (1.54 g, 11.5 mmol) and a catalytic amount of3-chloroperoxybenzoic acid. This mixture was heated overnight at reflux,cooled and filtered, washing the solids with chloroform. The filtratewas washed with 5% w/v sodium carbonate solution (3×30 ml), 0.1M sodiumthiosulphate solution (50 ml), brine (50 ml) and then dried andfiltered. The solvent was removed to leave a yellow solid which waschromatographed through silica gel, using 4:6 dichloromethane-lightpetroleum ether as eluant. The main fraction yielded thebis(trichloromethyl) derivative (14) (0.81 g, 91%), m.p. >180° C.(dec.).

l. 4-(Nitrophenyl)-2,9-bis(methoxycarbonyl)-1,10-phenanthroline (15) Thehexachloride (14) (1.00 g, 1.86 mmol) was dissolved in 98% sulphuricacid (2.5 ml) and heated to 90° C. under nitrogen for 2 h. The mixturewas then cooled in an ice-salt bath before carefully quenching intomethanol (6 ml). The solution was heated to reflux for 45 min beforecooling and quenching with crushed ice. The mixture was neutralised with7N sodium hydroxide solution and the off-white precipitate was collectedand dried before recrystallisation from methanol/chloroform to give thenitrodimethyl ester as a monohydrate (0.60 g, 77%).

m. 4-(Aminophenyl)2,9-bis(methoxycarbonyl)-1,10-phenanthroline (16) Thenitrodiester (15) (0.25 g, 0.6 mmol) was slurried in methanol (50 ml)and cyclohexene (5 ml) and 10% Pd/C (50 mg) added. The mixture washeated under reflux under nitrogen for 16 h before cooling and filteringthrough Celite. The solids were washed with hot chloroform (30 ml) andthe combined filtrate reduced to small bulk to yield a red solid. Theproduct was purified by chromatography through silica gel, using 2% v/vmethanol in chlorform as eluant. The product was obtained as themonohydrate (90 mg, 35%).

n. Conjugation of Acridine with the amine (16) The amine (16) (39 mg,0.1 mmol) in dry chloroform (2 ml) was heated with 9-chloroacridine (50mg, 2.35 mmol) at reflux for 5 h. Diethyl ether (25 ml) was added toprecipitate, as an orange crystalline solid, the target ester (17) (40mg, 65%).

o. Hydrolysis of the Ester (17) The ester (17) (25 mg) was heated indistilled water at reflux for 5 h. The solution was cooled to afford acrystalline solid which was collected and dried to afford the targetintercalating acid (12) (20 mg).

Preparation of the Intercalator (18)

p. 4-(4-Bromobutoxy)-2,9-dimethyl-1,10-phenanthroline (20)4-Hydroxy-2,9-dimethyl-1,10-phenanthroline (19) (2.0 g, 5.9 mmol) washeated in refluxing acetonitrile (50 ml) in the presence of1,4-dibromobutane (10 g) and potassium carbonate (2.0 g). After 10 h thesolution was filtered, the solvent removed and the residue trituratedwith chloroform-diethyl ether to afford the title compound (20) as asolid (2.5 g). This was used without further purification.

q. 4-(4-Bromobutoxy)-2,9-bis(trichloromethyl)-1,10-phenanthroline (21)To the ether (20) (0.4 g, 1.11 mmol)) in refluxing carbon tetrachloride(30 ml) was added N-chlorosuccinimide (98 mg, 7.33 mmol) and a catalyticamount of 3-chloroperoxybenzoic acid. After 12 h at reflux the solutionwas cooled, filtered and the solvent evaporated from the filtrate toleave a pale yellow residue which was chromatographed through silicagel, using 1:1 benzene:acetone as eluant, to give the title compound(0.50 g, 70%), m.p >150° C. (dec.). Found: C, 37.74; H, 2.32; N, 4.95;C₁₈ H₁₃ N₂ Cl₆ BrO requires C,38.20; H, 2.32; N, 4.95%.

r. 4-(4-Bromobutoxy)-1,10-phenanthroline-2,9-dicarboxylic Acid (22) Thehexachloride (21) (0.4 g, 0.7 mmol) was heated in aqueous acetic acid(1:4) (50 ml) at reflux. Portions of sodium acetate (3×350 mg,anhydrous) were added at 30 min intervals and heating continued for atotal of 12 h. The solution was filtered and the bulk of the solventremoved by evaporation under reduced pressure to leave a fine powderwhich was recystallised from aqueous tetrahydrofuran to give the acid(80 mg, 27%). M.p >180° C. (dec.). Found: C, 49.31; H, 3.68, N, 6.93.C₁₈ H₁₇ N₂ O₅ Br.H₂ O requires C, 49.45; H, 3.92; N, 6.91%.

s. 4-4-(N-Phenanthridinium)butyloxy)-1,10-phenanthroline-2,9-dicarboxylicAcid Bromide (18). The acid (22) (50 mg, 0.11 mmol) was heated undernitrogen in phenanthridine (150 mg) at its melting point whilst stirringwith a small spatula. After 20 minutes the mixture was cooled to roomtemperature and triturated with diethyl ether to afford a light brownsolid (40 mg). The solid was sparingly soluble in water. No molecularion could be observed in its mass spectrum.

EXAMPLE 1

Assay Procedure:

A sample of the europium (III) complex of the probe (2) (100 μl,1.75×10⁻⁵ M) was added to a solution of the target material (A) (200 μl,2.6×10⁻⁵ M) and Denhardt's solution (100 μl) and the volume made up to atotal of 1 ml with buffer solution (0.01 MM Tween 20, 1M NaCl, 0.1MHEPES). The solution was hybridised by heating to 42° C. for 3 h beforecooling to room temperature. To the solution was added the sensitiser (1ml, 1×10⁻⁵ M in buffer) and the solution kept at room temperature for 1hour before being serially diluted (up to ×448), with the buffer andmeasurements of luminescence made by irradiating at 290 nm and measuringthe delayed emission in the region around 615 nm. FIG. 2 shows theoutput of the assayed solution at ×56 dilution concentrations: Target,4.46×10⁻⁸ M, Probe: 1.56×10³¹ 8 M; Sensitiser: 8.9×10⁻⁸ M!. Thereference solution consisted of the probe polynucleotide andintercalator at the same concentration but in the presence of theunrelated target (B)

EXAMPLE 2

As a further test of the above assay procedure we used probe 3284 (SEQID No:1) (GAGATCAACGAGCAAGAATTTCTT) and 3 different targets ie. matched3288 (SEQ ID No:2) (GCTAAAGAAATTCTTGCTCGTTGATCTCCACT), mismatched 2638(SEQ ID No:3) (GATCATTCATGACATTTTAAAAATTACAGG) and one base pairdifferent 3287 (SEQ ID No:4) (GCTAAAGAAATTCTTGCTCGTTGACCTCCACT).

The same procedures for labelling, purifying and hybridisation wasfollowed as before. The results are shown below.

    ______________________________________                                        CONC.      3288          3287   2638                                          ______________________________________                                        4.1 × 10.sup.-6 M                                                                  28            15     0.8                                           8.2 × 10.sup.-7 M                                                                  9             1.52   0.27                                          8.2 × 10.sup.-8 M                                                                  1.12          0.05   <0.04                                         8.2 × 10.sup.-9 M                                                                  0.2           <0.02  <0.01                                         ______________________________________                                    

It can be seen that there is a clear distinction between the threeoligonucleotides ie. 3288 (matched), 3287 (one base pair mismatched) and2638 (mismatched). Not only is there discrimination between matched andmismatched oligonucleotides but also the one base pair differentoligonucleotide produced less counts than the matched oligonucleotide.This also occurred at varying dilutions. It can be seen that, atconcentrations approaching 4×10⁻⁹ M there is a significant differencebetween the three targets. The distinction improves with dilution.

EXAMPLE 3

Assay procedure using PCR products:

The synthesis of the europium labelled probe 3284 (SEQ ID No:1)(GAGATCAACGAGCAAGAATTTCTT) is as previously outlined. To test theG551D-europium (mutant specific) oligonucleotide probe, polymerase chainreaction (PCR) products were required.

1) Mutant sequence only, Exon 11 homozygote

2) Mutant and normal sequence, Exon 11 heterozygote

3) Normal sequence, Exon 11 wild type

4) Unrelated normal sequence, Exon 10 wild type ##STR1##

    ______________________________________                                        Reaction of PCR products                                                      Reaction Mix                                                                  ______________________________________                                        Exon 11    H.sub.2 O      2240 μl                                                     10X ARMS Buffer                                                                              400 μl                                                      1 mM dNTP's    400 μl                                                      50 μM 3108 primer                                                                         80 μl                                                       50 μM 3109 primer                                                                         80 μl                                                       Aliquot:       60 × 40 μl                                            Tube numbers:  1, 2, 3 (×20 each)                            Exon 10    H.sub.2 O      700 μl                                                      10X ARMS Buffer                                                                              125 μl                                                      1 mM dNTP's    125 μl                                                      50 μM 2090 primer                                                                         25 μl                                                       50 μM 2091 primer                                                                         25 μl                                                       Aliquot:       20 × 40 μl                                            Tube Number:   4 (×20)                                       ______________________________________                                    

DNA Dilutions

The template for the reaction will be product amplified previously. ThePCR product will be diluted so that approximately 10,000 copies areadded to each new reaction.

    ______________________________________                                        Exon 11 product:                                                              1.0032 × 10.sup.-5 (molecular weight) = 1.66561 × 10.sup.-19      g = 1 molecule                                                                6.023 × 10.sup.23 (Avogadro's number)                                   Exon 10 product:                                                              1.4356 × 10.sup.5 (molecular weight) = 2.36692 × 10.sup.-19 g     = 1 molecule                                                                  6.023 × 10.sup.23 (Avogadro's number)                                   GD/GD product: 8 ng · μl.sup.-1                                                    8 × 10.sup.-9 g = 4.8 × 10.sup.10                                 molecules/μl                                                               1.665161 × 10.sup.-19 g                                GD/+ product: 7 ng/μl.sup.-1                                                                7 × 10.sup.-9 g = 4.2 × 10.sup.10                                 molecules/μl                                                               1.665161 × 10.sup.-19 g                                +/+ (Ex11) product: 6 ng · μl.sup.-1                                               6 × 10.sup.-9 g = 3.6 × 10.sup.10                                 molecules/μl                                                               1.66561 × 10.sup.-19 g                                 +/+ (Ex10 product: 6 ng · μ.sup.-1                                                 6 × 10.sup.-9 g = 2.53 × 10.sup.10                                molecules/μl                                                               2.36692 × 10.sup.-19 g                                 ______________________________________                                    

Electrophoresis of PCR products

Following aliquots for each DNA sample were electrophoresed:

    ______________________________________                                        1.  10 μl pooled product                                                                        2.    5 μl pooled product                             3.  2 μl pooled product                                                                         4.    19 μl 1:10 pooled product                       5.  5 μl 1:10 pooled product                                                                    6.    2 μl 1:10 pooled product                        7   10 μl concentrated product                                                                  8.    5 μl concentrated product                       9   2 μl concentrated product                                                                   10.   10 μl 1:10 concentrated product                 11. 5 μl 1:10 concentrated product                                                              12.   2 μl 1:10 concentrated product                  ______________________________________                                    

By comparison of PCR product band intensity with band intensity of theox174/HaeIII molecular weight standards, the following estimates of theDNA concentration of the concentration products were made:

    ______________________________________                                        GD/GD  15 ng-μl.sup.-1                                                                     150 nM solution                                                                           0.66 μl in 100 μl = 1 × 10.sup.-9                                 M                                                 GD/+   10 ng-μl.sup.-1                                                                     100 nM solution                                                                           1.00 μl in 100 μl = 1 × 10.sup.-9                                 M                                                 +/+     8 ng-μl.sup.-1                                                                      80 nM solution                                                                           1.25 μl in 100 μl = 1 × 10.sup.-9                                 M                                                 (Ex11)                                                                        +/+     5 ng-μl.sup.-1                                                                      35 nM solution                                                                           2.66 μl in 100 μl = 1 × 10.sup.-9                                 M                                                 (Ex10)                                                                        ______________________________________                                    

The products were diluted as follows:

    ______________________________________                                        Stock                              10 .sup.10 molecular/μl                 1. 10 μl product                                                                      +      990 μl H.sub.2 O                                                                       =    10.sup.8 molecular/μl                   2. 10 μl (1.)                                                                         +      990 μl H.sub.2 O                                                                       =    10.sup.6 molecules/μl                   3. 10 μl (2.)                                                                         +      990 μl H.sub.2 O                                                                       =    10.sup.4 molecules/μl                   4. 100 μl (3.)                                                                        +      990 μl H.sub.2 O                                                                       =    10.sup.3 molecules/μl                   5 μl of corresponding DNA was added to each reaction mix                   GD/GD   2.4 × 10.sup.4 molecules                                        GD/+    2.1 × 10.sup.4 molecules                                        +/+ (Ex11)                                                                            1.8 × 10.sup.4 molecules                                        +/+ (Ex10)                                                                            1.3 × 10.sup.4 molecules                                        ______________________________________                                    

PCR Product Concentration, Purification and Washing

After amplification, PCR products were accordingly pooled, 20 μl ofpooled product was set aside for gel analysis. The remaining product wasapplied to microcon filtration membranes.

400 μl pooled product added to microcon--5000 rpm for 10 minutes

400 μl pooled product added to microcon--5000 rpm for 10 minutes

500 μl H₂ O added to microcon--5000 rpm for 5 minutes

500 μl H₂ O added to microcon--5000 rpm for 5 minutes

The DNA was eluted from the membrane by the addition of 180 μl H₂ O andcentrifugation at 3500 rpm for 3 minutes. 20 μl of the concentrationproduct was retained for gel analysis.

    ______________________________________                                        Results                                                                             -VE      GD/GD   EX10++   GC/+ EX11++                                   ______________________________________                                        INt   7.1      38.5    21.1     35   18.5                                     ______________________________________                                         (using the same probe 3284)                                              

EXAMPLE 4

A further example of the use of the lanthanide enhanced signallingsystem of the invention was obtained by hybridisation of theoligonucleotide probe 3922 (SEQ ID No:5) (GAGGTCAACGAGCAAGAATTTCTTGC) tothe oligonucleotide targets 3860 (SEQ ID No:6)(GCTAAAGAAATTCTTGCTCGTTGACCTCCACT) and 3288 (SEQ ID No:2)(GCTAAAGAAATTCTTGCTCGTTGATCTCCACT). Target 3860 comprises normal DNAsequence obtained from exon 11 of the CFTR gene which is unaffected bythe G551D mutation. Target 3288 is comprises CFTR exton 11 sequencecontaining the G551D mutation. The 5' 24 bases of probe 3922 arecomplementary to a sequence contained within target 3860. 23 of the 24bases at the 5' terminus of probe 3922 are complementary to target 3288but the guanosine residue at position 5'-4 is mis-matched to a thymidineresidue in that target.

The procedures for labelling, purification and hybridisation are asdescribed above. Hybridisation reactions were effected withequimolecular ratios of probe, target and intercalator andconcentrations of 2×10⁻⁷ M. A negative control hybridisation containedno target oligonucleotide. The results obtained are shown in FIGS. 3-5.The solutions were diluted to 3.1×10⁻⁹ M (i.e. ×64) and new readingswere obtained as shown in FIG. 6.

The data shows a clear discrimination between targets 3860 and 3288 byprobe 3922. A greater hybridisation signal is generated by probe 3922when it is perfectly matched to its target at its 5' terminus (3860)than when it is mis-matched (3288). Discrimination between targets 3860and 3288 improves in the 64× dilutions and background signal from the notarget control was undetectable at this dilution.

It should be noted that a further design feature of probe 3922 is thatits 3' terminus and penultimate bases are mis-matched to both targets3860 and 3286. These mismatches have been introduced into probe 3922 todestabilise the 3' terminus of that probe causing it to be refractory toamplification in the PCR or other gene amplification systems. Probe 3922may therefore be included in PCR reactions which have been designed toamplify exon 11 of the CFTR gene without the probe serving as a PCRprimer itself. Alternatively, blocking groups or dideoxynucleotides maybe included at the 3' terminus of such probes to prevent their extensionby Taq polymerase during the PCR.

The chemical formulae referred to in the application text above are asfollows: ##STR2##

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAGATCAACGAGCAAGAATTTCTT24                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCTAAAGAAATTCTTGCTCGTTGATCTCCACT32                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GATCATTCATGACATTTTAAAAATTACAGG30                                              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCTAAAGAAATTCTTGCTCGTTGACCTCCACT32                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GAGGTCAACGAGCAAGAATTTCTTGC26                                                  (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCTAAAGAAATTCTTGCTCGTTGACCTCCACT32                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GTGGTAATTTCTTTTATAGTAGAA24                                                    (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CACCATTAAAGAAAATATCATCTT24                                                    (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGGGAATCACCTTCTGTCTACAAT24                                                    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We claim:
 1. A method for detecting a nucleic acid analyte in a sample,which method comprises contacting the sample with (i) a binding entitycomplementary to the analyte and to which is attached a first partner ofa ligand to metal energy transfer system, (ii) a lanthanide ion, and(iii) a second partner of the system to which is attached a duplexbinder; wherein the first partner of the ligand to metal energy transfersystem is a complexing sensitiser and the second partner is a chelatingagent, or vice versa; such that upon complementary binding of theanalyte and the binding entity to form a nucleic acid duplex,interaction of the duplex binder with the duplex occurs and allows thefirst and the second partners of the system to form a closed chelatedsystem around the lanthanide ion and, upon irradiation, emission oflight from the lanthanide ion indicates the presence of the analyte inthe sample.
 2. A method as claimed in claim 1 wherein the duplex binderis selected from the group consisting of an intercalator and a groovebinder.
 3. A method as claimed in claim 1 wherein the binding entity isa polynucleotide to which the first or the second partner is attached.4. A method as claimed in claim 3 wherein the binding entity comprises ablocked terminal nucleotide.
 5. A method as claimed in any one of theprevious claims wherein the binding entity comprises one or moremismatched bases opposite a complementary analyte nucleic acid sequence.6. A method as claimed in claim 5 wherein a position of the one or moremismatched bases is selected such that duplex binding and henceformation of the closed chelated system around the lanthanide ion aremodulated.
 7. A method as claimed in claim 1 wherein the lanthanide ionis an europium ion.
 8. A method as claimed in claim 1 wherein thecomplexing sensitiser comprises an aromatic group.
 9. A method asclaimed in claim 1 wherein the complexing sensitiser comprises aheterocyclic aromatic group.
 10. A method as claimed in claim 1 whereinthe complexing sensitiser comprises an optionally substituted dipyridylor phenanthroline group.
 11. A method as claimed in claim 1 wherein thecomplexing sensitiser comprises an optionally substitutedphenanthroline-2,6-dicarboxylic acid group.
 12. A method as claimed inclaim 1 wherein the chelating agent is chelated with the lanthanide ionbefore addition to the assay medium.
 13. A method as claimed in claim 1wherein the binding entity is a polynucleotide to which a chelatedlanthanide ion is attached.
 14. A method as claimed in claim 1 whereinone partner of the ligand to metal energy transfer system is acomplexing sensitiser linked to a duplex binder.
 15. A method as claimedin claim 1 wherein the binding entity is an allele-specificpolynucleotide.
 16. A method as claimed in claim 1 which comprisesamplification of a target sequence to provide the nucleic acid analyte.17. A method as claimed in claim 16 wherein one or more components ofthe method are present during amplification of the target sequence. 18.A method as claimed in claim 1 wherein the binding entity is comprisedof two or more polynucleotide sequences.
 19. A method as claimed inclaim 18 wherein the first and the second partners of the ligand tometal energy transfer system are attached to different polynucleotidesequences.
 20. A method as claimed in claim 1 wherein the chelatingagent is a polyfunctional compound comprising carboxylic acid, amideand/or ether moieties.
 21. A method as claimed in claim 20 wherein thechelating agent is a derivative of a compound selected from the groupconsisting of ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA) andtrans-1,2-diaminocyclohexanetetraacetic acid (DCTA).
 22. A method asclaimed in claim 1 wherein a Forster energy transfer acceptor is used toreceive energy from the closed chelated system around the lanthanide ionand to emit light.
 23. A method as claimed in claim 22 wherein theForster energy transfer acceptor is bound to a polynucleotide.
 24. Amethod as claimed in claim 1 for detection of more than one nucleic acidanalyte in a sample.
 25. A method as claimed in claim 1 wherein thecharacteristics of light emission are measured to indicate the natureand/or quantity of the nucleic acid analyte.
 26. A method as claimed inclaim 1 which is a homogeneous assay.
 27. An assay kit which comprises(i) a binding entity complementary to the analyte and to which isattached a first partner of a ligand to metal energy transfer system,(ii) a lanthanide ion, (iii) a second partner of the system to which isattached a duplex binder; wherein the first partner of the ligand tometal energy transfer system is a complexing sensitiser and the secondpartner is a chelating agent, or vice versa; and further comprising oneor more of the following: buffer(s), amplification primers andinstructions for use of the kit.
 28. An assay kit as claimed in claim 27wherein the amplification primers are for allele specific amplification.29. A compound which comprises a duplex binder covalently linked to alanthanide ion sensitiser.
 30. A compound as claimed in claim 29 whichcomprises a phenanthroline-2,6-dicarboxylic acid group linked to aphenanthridine group.
 31. A compound as claimed in claim 30 of theformula ##STR3##
 32. A composition which comprises a duplex bindercovalently linked to a lanthanide ion chelating agent and a lanthanideion.
 33. A compound as claimed in any one of claims 27 and 29-31 whereinthe duplex binder is selected from the group consisting of anintercalator and a groove binder.